"A REFERENCE COMPOUND FOR USE IN THE ANALYSIS OF LEVOSIMEDAN BATCHES"

Abstract

The present invention relaes to a pyndazinyl derivative (I) and use as a reference compound in the Jetermination of potentially genotoxic impurities in levosimendan samples. The invention also relates to an analytic method for the determintion of potentially genotoxic impurities in levosimendan samples wherein (I) is used as a reference compound. Levosimendan is a medicanent useful in the treatment of heart failure.

Full Text

1
A REFERENCE COMPOUND FOR USE IN THE ANALYSIS OF LEVOSIMENDAN BATCHES
Background of the invention
The present invention relates to [[4-(2-azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyl]hydrazono]propanedinitrile (I) and its use as a reference compound in the analysis of batches of levosimendan synthesis, particularly in the determination of potentially genotoxic impurities in samples of a levosimendan batch. The present invention also relates to an analytic method for the determination of potentially genotoxic impurities in samples of a levosimendan batch wherein [[4-(2-azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyl]hydrazono ] propanedinitrile (I) is used as a reference compound.
Levosimendan, which is the (-)-enantiomer of [[4-(l,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propanedinitrile, and the method for its preparation is described e.g. in EP 565546 Bl. Levosimendan is potent in the treatment of heart failure and has significant calcium dependent binding to troponin. The use of levosimendan in the treatment of myocardial ischemia is described in WO 93/21921. The hemodynamic effects of levosimendan in man are described in Sundberg, S. et at, Am. J. Cardiol., 1995; 75: 1061-1066. Levosimendan is represented by the formula:

Clinical studies have confirmed the beneficial effects of levosimendan in heart failure patients.
Levosimendan can be prepared in high purity and in nearly quantitative yields by treating (-)-6-(4-aminophenyl)-5-methyl-4,5-
2
Summary of the invention
It has now been found that the potential genotoxic properties of fevosimendan samples are caused by an azido derivative impurity of formula (I)

This azido derivative is a potent matagen which can be formed curing levosimendan synthesis in an amount suff.;ient to give a positive result in the bacterial rmr.agenity test. This compound named [[4-(2-azido-3-methyI-5-oxotetrahydrofuran-2-yl)phcnyl]hydrazonc]propanedinitrilc (I) is therefore useful as a reference compound in the routine analysis of batches of levosimenda:. synthesis, particularly in the determination of potentially genotoxic impurities in levosimendan batches. The presence of (I) in a levosimer.dan batch indicates that further recrystallization is necessary for obtaining ievosimendan material suitable for use as a medicine.
Brief description of the drawings
FIG. 1 is a HPLC-chromatogram o: the [[4-(2-azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyI]hydrazono]propanedinitrilc standard (0.- g/ml).
FIG. 2 is a HPLC-chromatogram o: levosimendan raw material. Detailed description of the invention
The present invention provides a reference compound having structure (I) for the determination of potentially genotoxic impurities in samples of a levosimendan batch. The present invention also provides a reference preparation for the determination of potentially genotoxic impurities in samples of a levosirnendan batch comprising compound (I) optionally together with an analytically acceptable carrier, and in particular essentially in the absence of pyridazinyl derivatives.

3
The present invention also provides the use of (I) as a reference compound in the determination of potentially genotoxic impurities in samples of a levosimendan batch.
The present invention also provides an analytic method for the determination of potentially genotoxic impurities in samples of a levosimendan batch characterized in that compound (I) is used as a reference compound, particularly as said levosimendan sample is analyzed by High Pressure Liquid Chromatography (HPLC).
Furthermore, the present invention provides an analytic method for the determination of potentially genotoxic impurities in samples of a levosimendan batch which method comprises preparing a reference standard solution by dissolving compound (I) in solution, preparing a test solution by dissolving a sample of a levosimendan batch in solution, obtaining a HPLC chromatogram of said reference standard solution, obtaining a HPLC chromatogram of said test solution and determining the concentration of compound (I) in the sample.
The term "analytically acceptable carrier" means here a carrier which does not hamper the qualitative or quantitative analysis of (I). The selection of a suitable analytically acceptable carrier is well known to one skilled in the an. An example of an analytically acceptable carrier is dimethyl sulphoxide.
Compound (I) can be prepared by treating 6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone with sodium nitrite and malononitriie wherein sodium nitrite is used in molar excess and separating (I) from the reaction mixture. The procedure is described in detail in Example 1.
The product of a levosimendan batch is preferably analyzed using High Pressure Liquid Chromatography (HPLC). Suitable apparatus include e.g. C-8 reversed phase HPLC column with UV-detection at 360 nm. The mobile phase is e.g. a mixture of phosphate buffer pH 2.1 and acetonitrile. The levosimendan sample is dissolved in suitable solvent such as a mixture of dimethyl sulfoxide, methanol and water. The reference solutions are prepared by dissolving compound (I) in suitable solvent. The retention times of levosimendan and compound (I) or diastereomers thereof are determined in the chromatographic conditions used. The amount of (I) in the levosimendan sample is determined by comparing the chromatograms obtained for the sample solution and the reference solution. The procedure is described in detail in Example 2.

Ca = concentration of the major diastereomer of compound (I) in [[4-(2-azido-3-mcthyl-5-oxotetrahydro-furan-2-yl)phenyl]hydrazono]propane-dinitrile standard solution (mg/ml)
Rx = the peak area of the major diastereomer of compound (I) in the chromatogram of the test solution
Ra = the peak area of the major diastereomer of compound (I) in the
chromatogram of the [[4-(2-azido-3-methyl-5-oxotetrahydro-furan-2-yl)-phenyl3hydrazono]propanedinitrile standard solution
w = weight of the sample
10 = dilution factor of the sample (ml)

7
The concentration of the minor diastereomer of compound (I) in ppm will be calculated according to the following equation. The major diastereomer of compound (I) is used as a reference standard.

C51 = concentration of the minor diastereomer of compound (I) in [[4-(2-azido-3-methyl-5-oxotetrahydro-furan-2-yl)phenyl]hydrazono]propane-dinitrile standard solution (mg/ml)
Rx = the peak area of the minor diastereomer of compound (I) in the chromatogram of the test solution
Rst = the peak area of the minor diastereomer of compound (I) in the
chromatogram of the [[4-(2-azido-3-methyl-5-oxotetrahydro-furan-2-yl)-phenyl]hydrazono]propanedinitrile standard solution
w = weight of the sample
10 = dilution factor of the sample (ml)
1.023 = correction factor for the minor diastcrcomer of compound (I)
The chromatogram of the [[4-(2-azido-3-mcthyl-5-oxotetrahydro-furan-2-yl)phenyl]hydrazono]propanedinitrile standard (0.4 ug/ml) obtained is shown in Figure 1. The retention time for the major diastereomer of compound (I) is 25.180 min and for the minor diastereomer of compound (I) 24.866 min. The chromatogram of levosimendan raw material is shown in Figure 2.

8
WE CLAIM :

1.

A compound characterized by having the structure of formula I Q

and diastereomers thereof.
2. A reference preparation for the determination of potentially genotoxic impurities in
samples of a levosimendan batch comprising compound as claimed in claim 1.
3. A preparation as claimed in claim 2, essentially in the absence of pyridazinyl compounds.
4. A reference preparation for the determination of potentially genotoxic impurities
in samples of a levosimendan batch comprising compound as claimed in claiml together with an analytically acceptable carrier.
5. A preparation as claimed in claim 4, essentially in the absence of pyridazinyl compounds.
6. A compound as claimed in claiml, capable of being used as a reference compound
in the determination of potentially genotoxic impurities in samples of a
levosimendan batch.
7. An analytic method for the determination of potentially genotoxic impurities in
samples of a levosimendan batch characterized in that compund as claimed in claim 1
is used as a reference compound.
8. A method as claimed in claim 7, characterized in that a sample of a levosimendan batch is analyzed by High Pressure Liquid Chromatography (HPLC).

9
9. An analytic method for the determination of potentially genotoxic impurities in samples of a levosimendan batch which method comprises preparing a reference standard solution by dissolving compound as claimed in claim 1 in solution, preparing a test solution by dissolving a sample of a levosimendan batch in solution, obtaining a HPLC chromatogram of said reference standard solution, obtaining a HPLC chromatogram of said test solution and determining the concentration of compound as claimed in claim 1 in the sample.
The present invention relaes to a pyndazinyl derivative (I) and use as a reference compound in the Jetermination of potentially genotoxic impurities in levosimendan samples. The invention also relates to an analytic method for the determintion of potentially genotoxic impurities in levosimendan samples wherein (I) is used as a reference compound. Levosimendan is a medicanent useful in the treatment of heart failure.